The purpose of the investigations proposed in this application is to broaden our understanding of the biological role of the pteroylpolyglutamates. Support for the hypothesis that chain length of the polyglutamates serves as an element of regulation of one-carbon metabolism will be sought. Preliminary evidence indicates that separate pools of folate coenzymes committed to only certain of the folate requiring reactions exist, and that these pools differ from each other in the length of their poly-gamma-glutamyl chains as well as in the nature of their one-carbon substituents. To study this problem new methods based on the oxidation of folates, doubly labeled (2-14C,3'5'-9-3H) in vivo, and on the high pressure liquid chromatographic separation and identification of the resulting products, are being developed. These methods will allow the determination of chain length of pteroylpolyglutamates bearing one-carbon substituents of different oxidation levels. These procedures will be applied to the study of polyglutamate patterns of biological systems differing in the steady-state of their one-carbon metabolism either as a result of experimental manipulations or neoplastic transformation. The patterns of polyglutamates found in subcellular fractions will be studied. Also, the patterns observed in transplantable tumors of different degrees of malignancy, but originating from the same organ, will be determined and correlated to their respective rates of cell multiplications. The activity of conjugates (pteroyl-poly-gamma-glutamyl hydrolases) will be determined in all the experimental models used since these ubiquitous enzymes, for which no known biological role exists, are likely to be important modifiers of the naturally occurring folate coenzymes and, as such, likely to play a part in the regulation of one-carbon metabolism. The biosynthesis of pteroylpolyglutamates by enzyme preparations from avian liver will be investigated. Present knowledge of polyglutamate biosynthesis is limited and, in the context of hypothesis, necessitates further study.